The widespread dependence on antibiotics in medicine and agriculture have resulted in the continued emergence of antibiotic resistance, raising the specter of the end of the antibiotic era. This crisis has underscored the need for the identification of new antibiotics and anti-infective strategies. Our research proposal focuses on the exploration of an untapped potential reservoir of antimicrobials-compounds that we hypothesize nematodes employ as chemical defense against bacteria and fungi, in habitats characterized by highly complex microbiomes, including compost soil, rotting fruit, and decomposing insect carcasses. Our proposal brings together the highly complementary expertise from the field of Caenorhabditis elegans innate immunity and host-microbe interactions (D.K.), and from the field of C. elegans metabolomics and small- molecule signaling (F.S.). We will focus on metabolomic analysis of the nematode species C. elegans and Pristionchus pacificus, each of which feed on bacteria and are exposed to a wide variety of non-pathogenic and pathogenic bacteria and fungi in their natural environments, but which also exhibit differences in susceptibility to bacteria. Our recent studies of the metabolomes of these nematodes have identified several novel classes of small molecules of yet undetermined function that have chemical structures suggestive of interactions with bacteria. We propose to develop nematode metabolite libraries that are enriched for compounds produced by these nematode hosts in response to pathogenic bacteria and fungi, including the human pathogens Pseudomonas aeruginosa and Staphylococcus aureus. We will then screen these small molecule metabolite libraries for antimicrobial activity, taking advantage of established pathogenesis assays that follow microbial proliferation and survival of the nematode host, with subsequent definitive identification of active compounds via comparative metabolomics, and chemical synthesis. We anticipate finding compounds that directly affect bacterial and fungal viability as well as metabolites that function through the modulation of microbial colonization and virulence mechanisms. Our project has the potential to identify new classes of antimicrobial compounds and potentially novel anti-infective mechanisms that may help stem the tide of antibiotic resistant organisms.